Photoreceptors and bipolar cells of retina release neurotransmitter in a graded-continuous manner rather than in bursts in response to action potentials. To do so, these cells have evolved synaptic ribbons, proteinaceous structures that tether large numbers of synaptic vesicles near release sites. The molecular biology of the synaptic ribbon is poorly understood. A handful of proteins have been localized to the synaptic ribbon and the importance of these individual molecules as well as how they contribute to the unique functions of the synaptic ribbon remains elusive. Of these proteins, the most abundant is ribeye, a protein thought to constitute most of the synaptic ribbon and hypothesized to form the core of the synaptic ribbon. The precise role of ribeye remains unknown and the long-term goal of this proposal is to determine the functional role of ribeye in the synaptic ribbon. To do so, we will employ a combination of molecular biology, morphological analysis and electrophysiology using two model systems: zebra fish and mouse. In Aim 1 we investigate the property of ribeye to self-aggregate and be directed to the synapse using ribeye over expression mutants in zebra fish. The aggregation has been proposed to underlie the formation of the ribbon itself and thus may be critical in ribbon formation. Not only will these experiments inform us about ribeye localization, these experiments are essential to interpreting functional studies. In Aim 2, we intend to investigate the effects of over expression of wild-type and mutant ribeye on retinal responses to light using electroretinography. In preliminary experiments, we have identified transgenic lines that exhibit altered ERG b-wave responses. Any mutations that give rise to changes in the b-wave of the ERG will be further analyzed in aim 4 using whole-cell voltage clamp technique. In Aim 3, we will investigate the effect of ribeye mutant over expression on ribbon morphology. Since ribeye is the major constituent of the ribbon and may form the scaffold upon which the synaptic ribbon is built, over expression of ribeye or mutant versions of ribeye that lack important functional features of the protein may alter the morphology or number of synaptic ribbons. We will investigate the morphological features of transgenic animals generated in aim 1 using a combination of electron microscopy and stimulated emission-depletion microscopy (STED), a super-resolution light imaging technique. In Aim 4, we will evaluate the effects of ribeye mutants on synaptic release. To better understand the role of ribeye in synaptic transmission, we will investigate the effects of over expression of ribeye transgenes on synaptic release from rod bipolar cells on to AII amacrine cells in retinal slice recordings. The analogous mouse mutations of transgenes identified in aim 2 as having effects on b-wave of the ERG in zebra fish will be introduced into mouse rod bipolar cells by in vivo electroporation and then used for paired whole-cell recordings. Paired recordings will be used to determine the effects on vesicle pool size, rates of continuous release, recovery from depression and multivesicular release. Understanding synaptic ribbon function at the molecular level will ultimately aid in understanding how visual and auditory information is processed and communicated. In addition, it may provide clues to help understand diseases that specifically affect vision and hearing, such as Usher syndrome. In addition, the fundamental understanding of presynaptic processes in these specialized neurons will have broader implications for neuronal communication in general and thus, may contribute to our understanding of various aspects of mental health and neurological disorders. PUBLIC HEALTH RELEVANCE: Information in the nervous system is transmitted at the synapse via the release of neurotransmitter. In the retina and inner ear, primary sensory information is transmitted at specialized synapses specially evolved to transmit high rates of neurotransmitter release in a graded manner. We aim to understand, at the molecular level, how these cells accomplish this task. Understanding these synapses will ultimately aid in understanding how visual and auditory information is processed and communicated and provide clues to help understand diseases that specifically affect vision and hearing, such as Usher syndrome. In addition, the fundamental understanding of presynaptic processes in these specialized neurons will have broader implications for neuronal communication in general and thus, may contribute to our understanding of various aspects of mental health and neurological disorders.